Zebrafish scn1ba_tv1 and scn1ba_tv2 are splice variants. A. Upper panel: Comparison of Scn1ba_tv1, Scn1ba_tv2 and Scn1b amino acid sequences. Amino acid residues that are identical are indicted in red, strongly similar substitutions are indicated by (:), and weakly similar amino acids are indicated by (.). Identical resides in exon 5 of Scn1ba_tv1 and Scn1b are indicated in green. The two cysteine residues predicted to form the Ig loop are indicated in blue. The conserved regions that form the A/A' face of the Ig loop, sites of interaction with theα subunit , are underlined. Tyrosine-181 in Scn1b and the corresponding residues in Scn1ba_tv1 and Scn1ba_tv2 are highlighted in yellow. Predicted sites of N-linked glycosylation are indicated by ▼. These sites were determined using NetNGlyc 1.0 . Transmembrane segments are indicated as boxes. Peptides used for antibody generation are underlined in blue. Predicted β-sheets in the Ig loop domain, based on the crystal structure of myelin P0 , are shown with labeled arrows and correspond to the ribbon diagram included in the lower panel. Lower panel: Proposed three-dimensional structure of the Ig domain of β1 using the crystal structure of myelin Po (PDB 1NEU) as a template. The figure was created with the KiNG Viewer program via the RCSB Protein Data Bank web site . β strands corresponding to the arrows in the upper panel are labeled A through G. B. Schematic showing the genomic organization of zebrafish scn1ba. The positions of introns 1 through 5 (I1 – I5) are indicated. Positions of primers used for RT-PCR in panel D are indicated. The C-terminal alternate splice domains contained in scn1ba_tv1 and scn1ba_tv2 are encoded by exon 5. C. Model of alternative splicing of scn1ba. Exons 4 and 5 (boxes) and intron 4 (line) are illustrated. The splice acceptor sequence at the beginning of exon 5 is indicated by and the internal alternate splice acceptor site in exon 5 is indicated by a dashed line and by ▼. The location of stop codons in the resulting mRNAs are indicated. Drawings are not to scale. Consensus splice acceptor sequence  and the acceptor sequences found in exon 5 are indicated in the lower portion of the panel. Py: pyrimidine. Pu: purine. Lower case: intronic sequence. Upper case: exonic sequence. The "T" indicated by the red arrow in the internal, alternate acceptor is rare and significantly weakens the site . D. RT-PCR from whole fish RNA demonstrating that both splice variants of scn1ba are expressed in the mRNA pool. The upper band corresponds to scn1ba_tv1 and the lower band corresponds to scn1ba_tv2. Translations of the resulting alternate C-terminal splice products are shown below. The sequence highlighted in green is found in Scn1ba_tv1 and corresponds to the green portion of exon 5 illustrated in panel C. The sequence highlighted in turquoise is found in Scn1ba_tv2 and corresponds to the turquoise portion of exon 5 illustrated in panel C.